This invention is in the area of methods and compositions for the treatment of a host infected with hepatitis delta virus (also referred to as xe2x80x9cHDVxe2x80x9d) that includes administering an effective amount of a compound, in particular a nucleoside or nucleoside analog, that substantially reduces the level of hepatitis B surface antigen. In one nonlimiting embodiment, the nucleoside analog is 2xe2x80x2-fluoro-5-methyl-xcex2-L-arabinofuranosyl-uridine (also referred to as xe2x80x9cL-FMAUxe2x80x9d) or a pharmaceutically acceptable salt or prodrug thereof.
Type D hepatitis, the most severe form of viral hepatitis, is caused by infection with hepatitis D (delta) virus (HDV), a sub-viral satellite of hepatitis B virus (HBV) (Smedile, A., et al. (1994) Prog Liver Dis 12, 157-75). Compared with other agents of viral hepatitis, acute HDV infection is more often associated with fulminant hepatitis, a rapidly progressive, often fatal form of the disease in which massive amounts of the liver are destroyed. Chronic type D hepatitis is typically characterized by necroinflammatory lesions, similar to chronic HBV infection, but is more severe, and frequently progresses rapidly to cirrhosis and liver failure, accounting for the disproportionate association of chronic HDV infection with terminal liver disease (Smedile, A., et al. (1994) Prog Liver Dis12, 157-75; Rizzetto, M., et al. (1983) Ann Intern Med 98, 437-41). Although HDV infection affects fewer individuals than HBV alone, the resulting acute or chronic liver failure is a common indication for liver transplantation in Europe as well as North America (Smedile, A. and Rizzetto, M. (1992) Int J Clin Lab Res 22, 211-215; Wright, T. L. and Pereira, B. (1995) Liver Transplant Surgery 1, 30-42). Chronic disease affects 15 million persons worldwide, about 70,000 of whom are in the U.S. The Centers for Disease Control estimates 1,000 deaths annually in the U.S. due to HDV infection (Alter, M. J. and Hadler, S. C. (1993) Prog Clin Biol Res 382, 243-50; Alter, M. J. and Mast, E. E. (1994) Gastroenterol Clin North Am 23, 437-55).
There is currently no generally accepted effective therapy for type D hepatitis, and liver transplantation is the only option for the associated end-stage liver disease. Although interferon alpha has been moderately successful in treating some cases of type D hepatitis, the need for better treatment options is indicated by the very high doses required, variable responses, frequent relapse after cessation of treatment, and difficulties in drug administration (Thomas, H. C. et al. (1987) Prog Clin Biol Res 234, 277-90; Hoofnagle, J. et al. (1987) Prog Clin Biol Res 234, 291-8; Rosina, F. et al. (1987) Prog Clin Biol Res 234, 299-303; Rosina, F. et al. (1991) Hepatology 13, 1052-6; Farci, P. et al. (1994) N Engl J Med 330, 88-94; Hadziyannis, S. J. (1991) J. Hepatol 13 Suppl 1:S21-6; Di Marco, V. et al. (1996) J Viral Hepat 3, 123-8; Porres, J. C. et al. (1989) J Hepatol 9, 338-44).
Lamivudine (xcex2-L-2xe2x80x2,3xe2x80x2-dideoxy-3xe2x80x2-thiacytidine, 3TC) is a synthetic nucleoside shown to be effective in treating HIV and HBV infection. See U.S. Pat. No. 5,539,116 to Liotta et al. Lamivudine is known to cause sustained suppression of HBV replication during treatment (Nevens, F. et al. (1997) Gastroenterology 113:1258-1263). However, lamivudine does not improve disease activity or lower HDV-RNA levels in patients with chronic delta hepatitis (Lau, D. T.et al. (1999) Hepatology 30, 546-9). Lamivudine was recently approved in the U.S. and several other countries for treatment of chronic HBV infection. Prolonged treatment of chronic HBV carriers with lamivudine leads to decreased levels of HBV in serum and improved liver histology (Lai, C. L. et al. (1998) N Engl J Med 339, 61-8; Tyrrell, D. et al. (1993) Hepatology 18, 112A; Nevens, F. et al. (1997) Gastroenterology 113, 1258-63; Dienstag, J. L. et al. (1995) N Engl J Med 333, 1657-61). Despite the dramatic effects on HBV, lamivudine treatment of patients chronically infected with both HBV and HDV has little effect on circulating levels of HDV; more importantly, there is no improvement in disease activity even though HBV levels are suppressed (Honkoop, P. et al. (1997) Hepatology 24 (Suppl), 1219 (Abstract); Lau, D. T. et al. (1999) Hepatology 30, 546-9).
Additional forms of treatment have been tried. For example, suramin in vitro blocks the entry of the virion into hepatocytes, but it is too toxic to be acceptable for long term use in humans (Smedile, A., et al. (1994) Prog Liver Dis 12, 157-75). Acyclovir enhances HDV replication in vitro (Smedile, A., et al. (1994) Prog Liver Dis 12, 157-75). Ribavirin did not significantly affect virological or biochemical parameters and had severe side-effects (Smedile, A., et al. (1994) Prog Liver Dis 12, 157-75). Synthetic analogs of thymosin have also been ineffective in the treatment of HDV infection (Smedile, A. et al. (1994) Prog Liver Dis 12, 157-75).
None of the described treatments for HDV infection are generally accepted as effective. The HDV virion is composed of a ribonucleoprotein core and an envelope. The core contains HDV-RNA, and hepatitis delta antigen (HDAg), which is the only protein encoded by this virus (Wang, K. S. et al. (1986) Nature 323, 508-14). The envelope is formed by the surface antigen protein (hepatitis B surface antigen, or HBsAg) of the helper virus, hepatitis B. (Bonino, F. (1984) Infect Immun 43, 1000-5; Bonino, F. et al. (1981) Hepatology 1, 127-31; Bonino, F. et al. (1986) J Virol 58, 945-50). The envelope is the sole helper function provided by HBV. HDV is able to replicate its RNA within cells in the absence of HBV (Kuo, M. Y. et al. (1989) J Virol 63, 1945-50), but requires HBsAg for packaging and release of HDV virions (Wu, J. C. et al. (1991) J Virol 65, 1099-104; Ryu, W. S. et al. (1992) J Virol 66, 2310-2315.), as well as for infectivity (Sureau, C., et al. (1992) J Virol 66, 1241-5). As a result of the dependence of HDV on HBV, HDV infects individuals only in association with HBV.
Because the woodchuck hepatitis virus (WHV) is closely related to HBV (ca. 85% nucleic acid homology), it has been widely used as a model for HBV infection and disease in its natural host, the eastern woodchuck (M. monax) (Gerin, J. L. (1990) Gastroenterol Jpn 25 (Supp), 38-42; Tennant, B. C. et al. (1988) Viral Hepatitis and Liver Disease, 462-464). Experimentally infected woodchucks have also been used extensively for analysis and development of anti-HBV therapeutics. (Zahm, F. E. et al. (1998) Ital J Gastroenterol Hepatol 30, 510-6; Tennant, B. C. et al. (1998) Hepatology 28, 179-91; Mason, W. S. et al. (1998) Virology 245, 18-32; Korba, B. E. et al. (1996) Hepatology 23, 958-63; Hurwitz, S. et al. (1998) Antimicrob Agents Chemother 42, 2804-2809; Block, T. M. et al. (1998) Nat Med 4, 610-4; Cullen, J. M. et al. (1997) Antimicrob Agents Chemother 41, 2076-82; Fourel, G. et al. (1990) Nature 347, 294-8; Gangemi, J. et al. (1997) Antivir Therap 1, 64-70; Genovesi, E. V. et al. (1998) Antimicrob Agents Chemother 42, 3209-17; Korba, B. E. et al. (2000) Antiviral Res 45, 19-32; Korba, B. E. et al. (2000) Antiviral Therapy 55, 95-105; Korba, B. E. et al. (2000) Antimicrobial Agents and Chemotherapy 44, 19-32. The efficacy of several anti-HBV agents used to experimentally treat chronic WHV infection in woodchucks (araAMP, ribavirin, AZT, ACV, 3TC, famciclovir, FTC, alpha-interferon, fialuridine ganciclovir, thymosin alpha-1, combination therapy with 3TC and alpha-interferon or 3TC and famciclovir) has accurately paralleled the efficacy and_toxicity profiles of these agents administered to HBV patients treated in the course of clinical trials. The similar efficacy observed in WHV infected woodchucks and HBV infected persons treated with anti-HBV agents demonstrates that the woodchuck animal model can be predictive for anti-HBV therapies in man (Zahm, F. E. et al. (1998) Ital J Gastroenterol Hepatol 30, 510-6; Tennant, B. C. et al. (1998) Hepatology 28, 179-91; Mason, W. S. et al. (1998) Virology 245, 18-32; Hurwitz, S. et al. (1998) Antimicrob Agents Chemother 42, 2804-09; Fourel, G. et al. (1990) Nature 347, 294-8; Gangemi, J. et al. (1997) Antivir Therap 1, 64-70; Genovesi, E. V. et al. (1998) Antimicrob Agents Chemother 42, 3209-17; Korba, B. E. et al. (2000) Antiviral Res 44, 19-32; Korba, B. E. et al. (2000) Hepatology 31, 1165-75; Korba, B. E. et al. (2000) Antiviral Therapy 5, 95-105; Korba, B. E. et al. (2000) Antimicrob Agents Chemother 44, 1757-60). Like HBV, WHV can support HDV particle formation and infection, and the eastern woodchuck has been a useful model for HDV infection (Negro, F. et al. (1989) J Virol 63, 1612-8; Parana, R., Gerard, F., Lesbordes, J. L., Pichoud, C., Vitvitski, L., Lyra, L. G. and Trepo, C. (1995) J Hepatol 22, 468-73; Ciccaglione, A. R. et al. (1993) Arch Virol Suppl 8, 15-21; Bergmann, K. F. et al. (1989) J Immunol 143, 3714-21; Ponzetto, A. et al. (1984) Proc Natl Acad Sci USA 81, 2208-12; Ponzetto, A. et al. (1987) Prog Clin Biol Res 234, 37-46).
The dependence of HDV on its helper virus, HBV, could suggest that successful treatment of HDV infection would follow successful treatment of the supporting HBV infection. Unfortunately, this does not appear to be the case, as illustrated by recent results obtained with the drug lamivudine (Glaxo-Wellcome, Inc.) (Honkoop, P. et al. (1997) Hepatology 24 (Suppl), 1219 (Abstract); Lau, D. T. et al. (1999) Hepatology 30, 546-9). The lack of an effect of lamivudine on disease in HBV-HDV infected patients underscores the direct role of HDV in disease severity in such patients. Although lamivudine inhibits HBV and WHV replication, it does not affect the production of viral surface antigen (Lau, D. T. et al. (1999) Hepatology 30, 546-9; Doong, S. L. et al. (1991) Proc Natl Acad Sci USA 88, 8495-9; Korba et al. Hepatology, (2000) 31, 1165-75). The life cycle of HBV and other representatives of this family of viruses (for example, WHV) is unique in that the process of replicating genomic copies of the virus and the production of viral proteins (for example, HBV or WHV surface antigens) are differentially regulated (Ganem, D. 1996. Hepadnaviridae; In xe2x80x9cFields Virologyxe2x80x9d Fields B N, Knipe D M, Howley P, ed. Lippincott-Raven, Philadelphia, p. 2703-2737). Therefore, antiviral agents, such as synthetic nucleosides (for example, lamivudine) which target viral polymerases, may significantly inhibit HBV replication (for example, as measured by a reduction in viremia), but not affect the level of viral MRNA or viral protein production (for example, as measured by the levels of HBV surface antigen in plasma or serum). Given that the life cycle of HBV is unique in differentially regulating viral proteins and that HBsAg can be produced from a number of alternative transcripts, it has not been known to date what parameters are essential to achieving a therapeutic end point for HDV.
U.S. Pat. No. 5,747,044 discloses recombinantly produced immunogenic HDV polypeptides useful as vaccines.
U.S. Pat. No. 5,932,219 to Chiron discloses the entire genome of the hepatitis D virus, a family of cDNA replicas of the entire HDV genome, and teaches that portions of these cDNA sequences are useful as probes to diagnose the presence of virus in clinical samples. The patent also discloses proteins encoded by the cDNA that are useful in the production of vaccines. In particular, the ""219 patent discloses a vaccine for hepatitis D which incorporates the p24 and p27 viral polypeptides. U.S. Pat. No. 5,750,350 to Chiron claims a kit useful in the analysis of hepatitis D virus which includes a peptide encoded by ORF 5 of the HDV genome. U.S. Pat. No. 5,747,044 claims a recombinantly produced immunogenic particle which raises antibodies against HDV, wherein the particle includes an immunogenic polypeptide encoded within ORF 5 of the HDV nucleotide sequence or its complement.
U.S. Pat. No. 6,020,167 assigned to Medeva Holdings B. V. discloses a method for treating chronic hepatitis, and in particular, hepatitis B, that includes administering a composition containing antiHBsAg.
U.S. Pat. No. 5,770,584 discloses a method for treating hepatitis virus infection by administering alkyl lipids or alkyl lipid derivatives.
U.S. Pat. No. 4,619,896 discloses a process for unmasking delta antigen in the blood of an animal, that includes treating serum with a surfactant and optionally with an antibody-antigen dissociating agent. The blood derived delta antigen is used as a diagnostic agent in the detection and determination of different classes of antibodies to hepatitis D virus.
United States statutory invention registration H1,345 discloses a method for preventing or treating hepatitis virus by administering a protein-prenyl transferase inhibitor.
Sureau, et al., Production of Infectious Hepatitis Delta Virus In Vitro and Neutralization with Antibodies Directed against Hepatitis B Virus Pre-S Antigens, Journal of Virology, February 1992, p 1241-1245 discloses that HDV particles produced in vitro are infectious and that (i) infectious particles are coated with HBV envelope proteins that contain the pre-S1 and pre-S2 regions, (ii) epitopes of the pre-S1 and pre-S2 domains of HBV envelope proteins are exposed at the surface of HDV particles, and (iii) that antibodies directed against those epitopes have neutralizing activity against HDV.
The nucleoside analog L-FMAU [2xe2x80x2-fluoro-5-methyl-xcex2-L-arabinofuranosyl-uridine] is a known compound and has been shown to have significant antiviral activity against HBV replication in cell culture, and against the related duck hepatitis B virus in both cell culture and infected ducks (Aguesse-Gennon, S. et al. (1998) Antimicrob Agents Chemother 42, 369-76; Balakrishna Pai, S. et al. (1996) Antimicrob Agents Chemother 40, 380-6; Chu, C. K. et al. (1995) Antimicrob Agents Chemother 39, 979-81; Fu, L. et al. (1999) Biochem Pharmacol 57, 1351-9; Kotra, L. P. et al. (1997) J Med Chem 40, 3635-44; Kukhanova, M. et al. (1998) Biochem Pharmacol 55, 1181-7; Ma, T. et al. (1997) J Med Chem 40, 2750-4; Ma, T. et al. (1996) J Med Chem 39, 2835-43; Xu, A. S. et al. (1998) Biochem Pharmacol 55, 1611-9; Yao, G. Q. et al. (1996) Biochem Pharmacol 51, 941-7); Peek, S. et al. (2001) Hepatology 33, 254-66; Zhu, Y. et al. (2001) J. Virol 75, 311-22.
U.S. Pat. Nos. 5,587,362 and WO 95/20595 to Chu et al. disclose and claim L-FMAU and its pharmaceutical compositions for the treatment of HBV, and provides a detailed description of the synthesis of the compound. U.S. Pat. No. 5,567,688 to Chu et al. claims a method for the treatment of HBV using L-nucleosides including L-FMAU. U.S. Pat. No. 5,565,438 to Chu et al., claims a method to treat humans infected with Epstein-Barr virus (EBV) with L-FMAU. U.S. Pat. Nos. 5,808,040 and 5,753,789 disclose the use of L-FMAU to stabilize an oligonucleotide by including the compound at the 5xe2x80x2-terminus, 3xe2x80x2-terminus, or the interior of the oligonucleotide. WO 98/15375 discloses a method for the manufacture of L-FMAU.
L-FMAU has been shown to be a remarkably potent and fast-acting antiviral agent against WHV replication in chronically-infected woodchucks (Korba, B. et al. (1999) Antivir. Res. 41, A54; Chu, C. et al. (1998) in Therapiesfor viral hepatitis, eds. Schinazi, R. and Sommadossi, J. (International Medical Press, Atlanta), Vol. pp 303-12; Peek, S. F. et al. (1997) Hepatology 26, 425A(Abstract 1187); Peek, S. F. et al. (2001) Hepatology 33, 254-66; Zhu, Y. et al. (2001) J. Virol 75, 311-22. It has also been disclosed that L-FMAU induces suppression of WHV surface antigen in serum (Korba, B. et al. (1999) Antivir. Res. 41, A54; Chu, C. et al. (1998) in Therapies for viral hepatitis, eds. Schinazi, R. and Sommadossi, J. (International Medical Press, Atlanta), Vol. pp 303-12; Peek, S. B. et al. (1997) Hepatology 26, 425A and Peek, S. B. et al. (2000) Hepatology 33, 254-66. L-FMAU has been shown to have a favorable pharmacokinetic profile and sufficient oral bioavailability in rats and woodchucks that make it suitable for once daily administration (Wright, J. D. et al. (1995) Pharm Res 12, 1350-3; Wright, J. D. et al. (1996) Biopharm Drug Dispos 17, 197-207; Witcher, J. W. et al. (1997) Antimicrob Agents Chemother 41, 2184-7).
Because of the large number of persons infected with hepatitis delta virus, the devastating effects of hepatitis delta virus infection on the individual, and the lack of effective treatments, there is a critical need for new and effective methods and compositions for the treatment of hepatitis delta virus infection.
Therefore, it is an object of the present invention to provide methods and compositions for the treatment of a host, including a human, infected with hepatitis delta virus.
It is a further object of the present invention to provide a method for identifying compounds effective in the treatment of hepatitis delta virus infection.
It has been now been discovered that administration of a nucleoside or nucleoside analog or a prodrug or a pharmaceutically acceptable salt thereof that substantially reduces the level of hepatitis B surface antigen (referred to herein as HBsAg) in a host is useful in the treatment of hepatitis delta viral infection in that host. By substantial reduction of HBsAg in a host it is meant that the nucleoside or nucleoside analog reduces the hepatitis B surface antigen at least approximately 100-fold or more, and preferably, 200- or 500-fold relative to pretreatment values in vivo or in vitro, or to not more than 1, and preferably, 0.5 or 0.1 microgram per milliliter in vivo, as measured in serum or plasma using any appropriate standard immunoassays for example the commercial assay for human HBsAg (AUSZYME(trademark), Abbott Laboratories or that described for woodchuck hepatitis B surface antigen in: Viral Immunology 6:161.169; Cote, P. J., C. Roneker, K. Cass, F. Schodel, D. Peterson, B. Tennant, F. DeNoronha, and J. Gerin. 1993).
It was previously known that if a nucleoside or nucleoside analog does not significantly reduce the level of HBsAg in a hepatitis delta infected host, for example, 3TC (xcex2-L-2xe2x80x2,3xe2x80x2-dideoxy-3xe2x80x2-thiacytidine), then that nucleoside is not effective in the treatment of hepatitis delta virus. However, given that the life cycle of HBV is unique in differentially regulating viral proteins and that HBsAg can be produced from a number of alternative transcripts, it has not been known to date what parameters are essential to achieving a therapeutic end point for HDV, including whether reduction, as opposed to elimination, of HBsAg, by a nucleoside or nucleoside analog would translate into any therapeutic effect on HDV. Further, there previously existed no information on the degree of HBsAg suppression needed to achieve this desired outcome, as measured by serum concentrations or the length of treatment required for a sustained effect. Known nucleosides do not target all templates for HBsAg production and none are known to routinely suppress serum HBsAg levels. Finally, a host liver cell must be co-infected with both HBV or a hepadnavirus other than hepatitis B that supports HDV infection and hepatitis delta virus in order for HBsAg suppression to be effective against hepatitis delta virus formation, and the proportion of liver cells making HBsAg but not co-infected is not known in each case. Liver cells infected with HBV alone could contribute to the serum HBsAg levels, yet suppression of HBsAg in these cells would not impact on hepatitis delta virus formation.
It has now been established for the first time through the paradigm nucleoside, and a nonlimiting embodiment, L-FMAU, that if a nucleoside suppresses the production of HBsAg, so as to affect a substantial and sustained reduction in serum or plasma HBsAg levels, i.e., to approximately 100-fold or less than pretreatment values in vivo or in vitro, it will be useful in the treatment of hepatitis delta virus.
Therefore, in one aspect of the invention, a method for the treatment of HDV infected host, in particular a human, is provided that includes the administration of an effective amount of a nucleoside or nucleoside analog that reduces HBsAg in the infected host at least approximately 100-fold, and preferably 200- or 500- fold, or more relative to pretreatment values in vivo or in vitro; or to not more than approximately 1 microgram, or preferably 0.5 or 0.1 microgram, per milliliter, as measured in serum or plasma using standard immunoassays (such as the commercial assay for human HBsAg (AUSZYME(trademark), Abbott Laboratories) or that described for woodchuck hepatitis B surface antigen in: Viral Immunology 6:161.169; Cote, P. J., C. Roneker, K. Cass, F. Schodel, D. Peterson, B. Tennant, F. DeNoronha, and J. Gerin. 1993.
In an alternative embodiment, it has been now been discovered that administration of a nucleoside or nucleoside analog that substantially reduces the level of preS1 antigen in a host is useful in the treatment of hepatitis delta viral infection in that host. By substantial reduction of preS1 antigen in a host it is meant that the nucleoside or nucleoside analog reduces the hepatitis B surface antigen at least approximately 100-fold or more, and preferably, 200- or 500-fold relative to pretreatment values in vivo or in vitro, using any appropriate assay, including that in: Deepen R, Heermann K H, Uy A, Thomssen R, Gerlich W H. xe2x80x9cAssay of preS epitopes and preS1 antibody in hepatitis B virus carriers and immune personsxe2x80x9d Med Microbiol Immunol (Berl). 1990;179(1):49-60.
In another aspect, a method for the treatment of an HDV infected host, in particular a human, is provided that includes the administration of an effective amount of an organic non-nucleoside small molecule (i.e., a molecule of molecular weight less than 500, which is other than a biologic material found in nature or a derivative or analog thereof retaining the desired activity, such as a peptide, protein, antibody, hormone, ribozyme, nucleic acid, or cytokine), and which is not a protein-prenyl transferase inhibitor or thymosin-alpha-1, that reduces HBsAg in the infected host to at least approximately 100-fold or more, and preferably at least 200- or 500-fold, relative to pretreatment values in vivo or in vitro; or to not more than 1 microgram, preferably 0.5 or 0.1 microgram per milliliter, as measured in serum or plasma using any appropriate assay, including standard immunoassays (such as the commercial assay for human HBsAg (AUSZYME(trademark), Abbott Laboratories) or that described for woodchuck hepatitis B surface antigen in: Viral Immunology 6:161.169; Cote, P. J., C. Roneker, K. Cass, F. Schodel, D. Peterson, B. Tennant, F. DeNoronha, and J. Gerin. 1993.
In one embodiment, an effective amount of L-FMAU or a pharmaceutically acceptable salt or prodrug thereof is administered to a host in need thereof to treat a hepatitis delta viral infection. In a preferred embodiment L-FMAU is administered to a host in need thereof in the absence of its corresponding xcex2-D enantiomer (i.e., in an enantiomerically enriched or enantiomerically pure form).
In another embodiment, L-FMAU is administered in combination with at least one other HBsAg or preS1 antigen lowering agent that can be another nucleoside, a nucleoside analog or a non-nucleoside for the treatment of a hepatitis delta infected host. In yet another embodiment, the hepatitis delta treating agent is administered in combination with a agent that has activity against hepatitis B, whether or not the anti-hepatitis B agent lowers the hepatitis B surface antigen.
In yet another embodiment, a method for treating hepatitis delta is provided that includes administering a compound, including a nucleoside, nucleoside analog or other small molecule as defined herein, that reduces the surface antigen of a hepadnavirus other than hepatitis B that supports HDV infection.
In another embodiment, a method for screening a compound, including a nucleoside or a nucleoside analog, that is effective in the treatment of HDV infection is provided that includes assessing whether the compound suppresses the expression of hepatitis B surface antigen 100-fold or more (and preferably 200- or 500-fold) relative to pretreatment values in vivo or in vitro; and preferably, to not more than 1 microgram (and preferably 0.5 or 0.1 microgram) per milliliter, as measured in serum or plasma using standard immunoassays (such as the commercial assay for human HBsAg (AUSZYME(trademark), Abbott Laboratories) or that described for woodchuck hepatitis B surface antigen in: Viral Immunology 6:161.169; Cote, P. J., C. Roneker, K. Cass, F. Schodel, D. Peterson, B. Tennant, F. DeNoronha, and J. Gerin. 1993, using methods provided herein or otherwise available.
In another embodiment, a method for screening a compound, including a nucleoside or a nucleoside analog, that is effective in the treatment of HDV infection is provided that includes assessing whether the compound suppresses the expression of hepatitis B surface antigen 100-fold or more (and preferably 200- or 500-fold) relative to pretreatment values in vivo or in vitro; or to not more than 1 microgram (and preferably 0.5 or 0.1 microgram) per milliliter, as measured in serum or plasma using standard immunoassays (such as the commercial assay for human HBsAg (AUSZYME(trademark), Abbott Laboratories) or that described for woodchuck hepatitis B surface antigen in: Viral Immunology 6:161.169; Cote, P. J., C. Roneker, K. Cass, F. Schodel, D. Peterson, B. Tennant, F. DeNoronha, and J. Gerin. 1993, using methods provided herein or otherwise available.
In another embodiment, a method for screening a compound, including a nucleoside or a nucleoside analog, that is effective in the treatment of HDV infection is provided that includes assessing whether the compound suppresses the expression of preS1 surface antigen 100-fold or more (and preferably 200- or 500-fold) relative to pretreatment values as measured in serum or plasma using standard immunoassays.
The invention is based on the fundamental discovery that L-FMAU, as the model active compound, suppresses the expression of hepatitis B surface antigen by a sufficient amount that it effectively inhibits the packaging and release of HDV virions. Evidence is presented herein that L-FMAU causes a substantial decrease in levels of HDV viremia by suppressing the expression of hepatitis B surface antigen.
In another embodiment, HDV infection can be treated in a host by administering at least one antisense oligonucleotide targeted to the RNA transcript of the hepatitis B surface antigen or preS1 antigen either alone or in combination with L-FMAU, or another HBsAg or preS1 antigen lowering nucleoside or nucleoside analog. The term xe2x80x9coligonucleotidexe2x80x9d encompasses both oligomeric nucleic acid moieties of the type found in nature, such as the deoxyribonucleotide and ribonucleotide structures of DNA and RNA, and man-made analogues that are capable of binding to nucleic acids found in nature. The oligonucleotides of the present invention can be based upon ribonucleotide or deoxyribonucleotide monomers linked by phosphodiester bonds, or by analogues linked by methyl phosphonate, phosphorothioate, phosphoroamidate, phosphorodithioate, or other oligonucleotide stabilizing bonds. They may also comprise monomer moieties which have altered base structures or other modifications, but which still retain the ability to bind to naturally occurring DNA and RNA structures. Such oligonucleotides may be prepared by methods well-known in the art, for instance using commercially available machines and reagents available from Perkin-Elmer/Applied Biosystems (Foster City, Calif.).
It is preferred that an antisense oligonucleotide targeting the HBV surface antigen or preS1 gene sequence be chosen such that the oligonucleotide hybridizes within approximately 25 bases of the AUG start codon of the gene. Examples of antisense oligonucleotides directed to the HBV surface antigen and preS1 gene are described in U.S. Pat. No. 5,646,262 to Korba et al. and include (SEQ ID NO.: 1) CTTAGGACTACACTACAAGAG; (SEQ ID NO.: 2) GACTACACTACAAGAG; (SEQ ID NO.: 3) AGGACTACACTACAAGAGGTA; (SEQ ID NO.: 4) TACACTACAAGAGGTA; (SEQ ID NO.: 5) TCTTCCCCAGGATCCT; (SEQ ID NO.: 6) TTTGGGGCGGACATTG; (SEQ ID NO.: 7) CCTAAGAACAGTTGTT; (SEQ ID NO.: 8) GTACAAGTCGCGTCCCAGG; (SEQ ID NO.: 9) TAGGAGCTCTTCTAAC; (SEQ ID NO.: 10) TATTCCCTAGTCTTGT; (SEQ ID NO.: 11) CAAGAGGTACAAGTC; (SEQ ID NO.: 12) CGACCACCTTTCTAAGACGGG; (SEQ ID NO.: 13) CCTTTCTAAGACGGG; (SEQ ID NO.: 14) TAAGACGGGGTA; (SEQ ID NO.: 15) GACGGGGTACGACAT; (SEQ ID NO.: 16) GTACGACATCTAGAA. Other examples of antisense oligonucleotides for the treatment of HDV infection are disclosed in U.S. Pat. No. 5,985,662 to Isis Pharmaceuticals, Inc. and include: (SEQ ID NO.: 17) CCTGATGTGATGTTCTCCAT; (SEQ ID NO.: 18) GAACTGGAGCCACCAGCAGG; (SEQ ID NO.: 19) GAAAGATTCGTCCCCATGC; and (SEQ ID NO.: 20) CCACTGCATGGCCTGAGGATG.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.